1. Field of the Invention
The present invention relates to a fluorescence detection apparatus which detects a fluorescent signal emitted from a specific substance contained in a sample, and which determines the amount of the substance based on the amount of the detected fluorescent signal, and more particularly to a fluorescence detection apparatus which is useful in the case where real time monitoring (tracking of a temporal change in the amount of a fluorescent signal) of a large number of samples is performed in the field of clinical diagnosis requiring incubation at a predetermined temperature, such as an enzyme reaction.
2. Description of the Related Art
The manner of producing a fluorescent reaction product due to an enzyme reaction may be monitored in real time. In such a case, fluorescence detection must be performed while incubating a sample (reaction solution) at a predetermined temperature. In the field of clinical diagnosis and the like, moreover, a large number of samples must be promptly processed at the same time.
In a first one of methods which are conventionally used in the field of clinical diagnosis and the like, fluorescence detection is performed while samples are transported along a guide the temperature of which is adjusted. For example, the temperature of a guide which is made of a material of excellent thermal conductivity such as an aluminum alloy is adjusted by means of a heater or the like. One or plural samples placed on a holder are transported along the guide by using a chain, a turntable, or the like. Fluorescent signals are sequentially detected by a fluorescence detector which is placed along the guide.
Another method is known in which connection type sample vessels, titer plates, or the like that can house a large number of samples are placed on temperature adjusting means, so that fluorescence detection is simultaneously performed on the large number of samples. A fluorescence detection apparatus which is useful in this case is characterized in that the apparatus is provided with (a) plural optical sensors or (b) a multichannel optical sensor, or has mechanical moving means for moving an optical sensor or a light guide (means for guiding a fluorescent signal emitted from a sample vessel to an optical sensor, such as an optical fiber).
The apparatus of (a) is a fluorescence detection apparatus in which optical sensors the number of which corresponds to that of samples to be simultaneously subjected to fluorescence detection are used and fluorescent signals emitted from the samples are independently detected. Such an apparatus usually has a configuration wherein a light guide for splitting excitation light from a light source and then guiding the split excitation light to samples is used.
In the apparatus of (b), an image sensor such as a CCD or a photodiode array is used in place of the plural optical sensors of (a). According to this configuration, fluorescent signals emitted from the arranged samples are detected as an image under a state where positional relationships among luminescent points are maintained. Also in such an apparatus, a configuration is usually employed in which excitation light from a single light source is guided to samples by using a split type light guide (an optical device, an optical fiber, or the like).
In the apparatus of (c), the optical sensor is mechanically moved over a large number of samples or the samples are sequentially moved to a fluorescence detection position for the optical sensor. In such an apparatus, the configuration wherein a light guide is mechanically moved is used most commonly. In this configuration, an excitation light guide and a fluorescence light guide are used, ends of the guides which are on the side of the samples are integrated with each other, and the guides are then simultaneously moved, whereby fluorescence is detected while sequentially exciting the large number of samples.
When such a conventional fluorescence detection apparatus is used for monitoring in real time a temporal change in a fluorescent signal emitted from a specific substance contained in a sample while incubating the sample at a predetermined temperature, there arise the following problems.
In the first method, the samples are transported along the temperature-adjusted guide, and then sequentially subjected to fluorescence detection. Consequently, there are problems such as an insufficient accuracy of the temperature adjustment, a limited number of processable samples, and a possibility of carryover. Specifically, it is difficult to adjust the whole of the transport guide so as to have a uniform temperature, and the thermal conductivity between the transport guide and the samples is hardly maintained constant over the whole of the guide. As a result, the temperatures of the samples may be changed during transportation, or the samples may be different in temperature from one another. Furthermore, the transported samples are subjected to fluorescence detection one by one. When the temporal change in a fluorescent signal is to be monitored for a long period, the same sample must be repeatedly transported. Therefore, the number of processable samples has its limit. Moreover, the possibility of contamination (carryover) among the samples due to splashes of the samples cannot be eliminated.
In the second method, the problems of the first method can be solved, but the following further problems may be produced.
In (a), plural optical sensors must be disposed. Therefore, the production cost is increased, and a space which corresponds to the number of the optical sensors is required. When the apparatus is to be miniaturized, the limitation on the space enables only several optical sensors to be disposed. As a result, the number of samples which can be simultaneously processed remains to be small. It may be contemplated that optical sensors of a small size such as photodiodes are used. However, such optical sensors have a drawback that the sensitivity to weak fluorescence is insufficient. Moreover, the sensitivity of each photodiode must be corrected. There is a further problem that the intensity of a fluorescent signal is proportional to that of the excitation light and hence splitting of the excitation light from the light source causes deterioration of the detection sensitivity.
In (b), sensitivity to weak fluorescence is insufficient. Therefore, (b) is not suitable for detection of weak fluorescence. In order to compensate the insufficient sensitivity, a measure such as a device (so-called image intensifier) which amplifies the amount of light by means of electronic amplification using a microchannel plate is sometimes further employed. In this case, however, the cost is very high. At present, therefore, such a measure is employed only in a special study purpose. Since fluorescence emitted from a wide area is detected as an image, there arise further problems that the amount of light is unevenly detected because of lens aberration, and that the burden of the data processing is increased by the extreme amount of data.
In (c), because of the limitation of the bendability of the light guide, the movement range is restricted, and there is a possibility that the light guide is broken. In the light guide, moreover, the light transmission efficiency is changed by bending, and hence fluorescence detection is hardly performed with high reproducibility. On the other hand, also the mechanical movement of the optical sensor involves the movement of a cable and other components, and hence has problems that the movement range is restricted, and that there is a possibility of breaking the cable or the like.
As described above, a fluorescence detection apparatus for monitoring a fluorescent signal in real time, and particularly that for performing monitoring in real time while incubating samples at a predetermined temperature must satisfy requirements such as (a) highly accurate temperature adjustment, (b) prompt treatment of a large number of samples, (c) high sensitivity, (d) high reliability (reduction in number of mechanical troubles typified by a cable breakage and malfunction of movable parts, improvement of reproducibility of fluorescence detection, and reduction of probability of carryover), (e) low cost (simplification of the configuration of the apparatus, and nonuse of expensive components in data processing and the like), and miniaturization of the apparatus.
Therefore, it is an object of the invention to provide a fluorescence detection apparatus which can satisfy the above requirements.
In order to attain the object, according to the invention, there is provided a fluorescence detection apparatus which detects a fluorescent signal emitted from a specific substance of a sample taken in a sample vessel, the apparatus comprising: a sample holder which fixedly holds sample vessels arranged on a same arc; a partition plate; a light guide which is configured by optical fibers and which transmits fluorescent signals emitted from respective test samples, to an optical sensor; the single optical sensor; and a light source which generates excitation light, wherein fluorescent signal emission ends of the light guide are opposed to the optical sensor, and fluorescent signal incidence ends of the light guide are respectively opposed to the sample vessels via the partition plate therebetween, wherein the partition plate includes excitation light optical means for selectively guiding the excitation light from the light source to only one of the sample vessels arranged on the arc, and fluorescence optical means for guiding only the fluorescent signal emitted from the selected one of the sample vessels to the light guide, and wherein the partition plate is coupled together with the excitation light optical means and the fluorescence optical means to driving means, to be rotatable about a center of the arc on which the sample vessels are arranged, and fluorescence is detected while the excitation light is guided sequentially to the sample vessels arranged on the arc, by rotation of the partition plate.